Inhibition of thymine DNA glycosylase in the treatment of cancer

ABSTRACT

The invention provides compositions, kits, and methods for inducing growth arrest, differentiation, or senescence of cancer cells that express thymine DNA glycosylase, and treating the cancer accordingly. The methods comprise inhibiting expression or biologic activity of thymine DNA glycosylase in cancer cells. Inhibition of thymine DNA glycosylase in cancer cells may induce the cells to revert to a healthy, non-cancerous phenotype and/or may induce the cells to senesce. Cancer cells include melanoma, lung, prostate, pancreatic, ovarian, brain, colon, recto-sigmoid colon, and breast cancer cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT international applicationnumber PCT/US14/58240, filed on Sep. 30, 2014, and claims priority toU.S. Provisional Application No. 61/884,478, filed on Sep. 30, 2013. Thecontents of each application are incorporated by reference herein, intheir entirety and for all purposes.

REFERENCE TO GOVERNMENT GRANTS

The invention was made with government support under Grant No. CA078412awarded by the National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named TDG Inhibitors ST25.txt, created on Sep. 10, 2014,with a size of 5898 bytes. The Sequence Listing is incorporated byreference herein.

FIELD OF THE INVENTION

The invention relates generally to the field of cancer treatment. Moreparticularly, the invention relates to inhibiting the expression orbiologic activity of thymine DNA glycosylase (TDG) in cancer cells ssuch as melanoma cells, lung cancer cells, prostate cancer cells, coloncancer cells, recto-sigmoid colon cancer cells, pancreatic cancer cells,ovarian cancer cells, and breast cancer cells and, thereby reducingproliferation and/or cell growth and/or inducing differentiation and/orinducing senescence of the cancer cells.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety and for all purposes.

Melanoma is an aggressive cancer that derives from the malignanttransformation of melanocytes, the pigment-producing cells that residein the basal layer of the epidermis in the skin, and in other organs,including the eye and the intestine. Melanomas are caused by genetic andepigenetic alterations in melanocytes affecting MAP kinase pathway(RAS-RAF), PTEN-AKT axis, p16INK4 (regulation of senescence), and MITF.Although targeted therapy, e.g. using RAF inhibitors, has improved theclinical management of melanoma for fifty percent of the patients for alimited period (6 months), an effective treatment of melanoma is stilllacking for the entire population and for a longer period of time.

SUMMARY OF THE INVENTION

The invention features methods for inhibiting the growth of premalignantor cancer cells in which TDG is expressed, methods for inducingdifferentiation of premalignant or cancer cells in which thymine DNAglycosylase (TDG) is expressed, and methods for inducing senescence inpremalignant or cancer cells in which TDG is expressed. In general, themethods comprise inhibiting the expression or inhibiting the biologicactivity of TDG in the premalignant or cancer cell. In some aspects,inhibiting the expression or the biologic activity of TDG in thepremalignant or cancer cell inhibits the growth of the premalignant cellor cancer cell. In some aspects, inhibiting the expression or thebiologic activity of TDG in the premalignant or cancer cell inducesdifferentiation of the cancer cell or premalignant cell. In someaspects, inhibiting the expression or the biologic activity of TDG inthe premalignant or cancer cell inhibits the growth of the cancer cellor premalignant cell and induces differentiation of the cancer cell orpremalignant cell. In some aspects, inhibiting the expression or thebiologic activity of TDG in the premalignant cell or cancer cell inducessenescence in the cancer cell or premalignant cell. The premalignant orcancer cell may express a high level, an intermediate level, or a lowlevel of thymine DNA glycosylase. Differentiation may comprise reversionof the cancer cell from a cancerous phenotype to a healthy phenotype.This reversion may include or otherwise be characterized, at least inpart, by morphologic changes in the cell. The morphologic changes mayinclude the loss of a spindle shape, and/or the acquisition of cellularprocesses emanating from the cell body. The morphologic changes maycomprise characteristic of the morphology of a melanocyte, anoligodendrocyte, a neuron, or an astrocyte. The cancer cell may be anycancer cell in which TDG is expressed or in which TDG is a factor in thetransformation of a healthy cell to a cancerous state or in which TDG isa factor in the progression of cancer. The cancer cell may be a melanomacell, colon cancer cell, recto-sigmoid colon cancer cell, prostatecancer cell, pancreatic cancer cell, ovarian cancer cell, breast cancercell, lung cancer cell, or brain cancer cell such as a glioblastomacell. The premalignant cell may be a skin cell that may progress to amelanoma cell, a colon cell that may progress to a colon cancer cell, arectal cell that may progress to a recto-sigmoid colon cancer cell, aprostate gland cell that may progress to a prostate cancer cell, anovary cell that may progress to an ovarian cancer cell, a pancreas cellthat may progress to a pancreatic cancer cell, a breast cell that mayprogress to a breast cancer cell, a lung cell that may progress to alung cancer cell, or a brain cell that may progress to a brain cancercell such as a glioblastoma cell.

The invention also features methods for treating cancer in a subject inneed thereof. The cancer preferably is a cancer in which TDG isexpressed. The methods generally comprise administering to a subjecthaving a cancer an effective amount of an agent that inhibits theexpression of TDG or an effective amount of an agent that inhibitsbiologic activity of TDG. Administration may comprise localized ordirect administration to the cancer, or may comprise systemicadministration, for example, to the blood of the patient. The subjectmay be any animal, and preferably is a human being. Non-limitingexamples of cancer that may be treated according to this method comprisemelanoma, colon cancer, recto-sigmoid colon cancer, prostate cancer,ovarian cancer, pancreatic cancer, breast cancer, lung cancer, and braincancer, including glioblastoma.

Inhibiting the expression of thymine DNA glycosylase may comprisetransfecting the cancer cell with a nucleic acid molecule thatinterferes with the expression of thymine DNA glycosylase. The nucleicacid molecule may comprise or encode a shRNA that specificallyhybridizes under stringent conditions to mRNA encoding thymine DNAglycosylase. The nucleic acid molecule may comprise or encode a shRNAthat specifically hybridizes under stringent conditions to mRNA encodinghuman thymine DNA glycosylase. The shRNA may comprise the nucleic acidsequence of SEQ ID NO: 3 or SEQ ID NO: 4, and/or may specificallyhybridize to the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5, or SEQ ID NO: 6, or the complement thereof. Transfecting the cancercell may comprise infecting the cell with a virus encoding the nucleicacid molecule that interferes with the expression of thymine DNAglycosylase. The virus preferably is a lentivirus.

Inhibiting biologic activity of TDG may comprise contacting a tumor cellwith an effective amount of an agent that inhibits the biologic activityof TDG, for example, via administration of the agent to a subject. Theagent may comprise an organic or inorganic chemical (including acomposition comprising such an organic or inorganic chemical, includinga small molecule, and a carrier such as a pharmaceutically acceptablecarrier) that inhibits the biologic activity of TDG. The agent maycomprise a biomolecule, including an antibody that specifically binds toTDG, or a polypeptide. The agent may comprise one or more of6-keto-prostaglandin F1a, prostaglandin A1, E6 berbamine, juglone,GW-5074, rottlerin, cefixime, idarubicin, doxorubicin, methenamine,Congo red, sodium ferric gluconate, ferrous sulfate, aurothioglucose,Evans blue, closantel, cinchonine sulfate, hexadimethrine bromide,indigotindisulfonate, and protamine chloride, or any combinationthereof. In some preferred aspects, the agent comprises juglone. In somepreferred aspects, the agent comprises cefixime. In some preferredaspects, the agent comprises closantel. The tumor cell may comprise amelanoma cell, colon cancer cell, recto-sigmoid colon cancer cell,prostate cancer cell, breast cancer cell, pancreatic cancer cell,ovarian cancer cell, lung cancer cell, or brain cancer cell.

In some aspects, any method may further comprise contacting the m tumorcell with an effective amount of one or more of a RAD51 inhibitor, a DNAalkylating agent, temozolomide, dacarbazine, cisplatin, vincristine, orany combination thereof, for example, via administration to a subject.

The invention also features kits. The kits may comprise an agent thatinhibits the expression of TDG, and/or an agent that inhibits biologicactivity of TDG, and instructions for using the agent in a method fortreating cancer, or in a method for inhibiting the growth of cancercells, or in a method for inducing differentiation of cancer cells, orin a method for inducing senescence in cancer cells. Such methods may beany methods described or exemplified herein.

The TDG expression-inhibiting agent may comprise or encode shRNA thatspecifically hybridizes under stringent conditions to mRNA encodingthymine DNA glycosylase. The shRNA may comprise the nucleic acidsequence of SEQ ID NO: 3 or SEQ ID NO: 4, and/or may specificallyhybridize to the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:5, or SEQ ID NO: 6, or the complement thereof, or to the nucleic acidsequence corresponding to nucleotides 892-912 of SEQ ID NO: 7, or thecomplement thereof. The TDG expression-inhibiting agent may comprise avirus encoding the shRNA. The virus preferably is a lentivirus. The TDGbiologic activity-inhibiting agent may comprise 6-keto-prostaglandinF1a, prostaglandin A1, E6 berbamine, juglone, GW-5074, rottlerin,cefixime, idarubicin, doxorubicin, methenamine, Congo red, sodium ferricgluconate, ferrous sulfate, aurothioglucose, Evans blue, closantel,cinchonine sulfate, hexadimethrine bromide, indigotindisulfonate,protamine chloride, or any combination thereof. Juglone, closantel, andcefixime are preferred. The agent may be comprised in a composition witha carrier such as a pharmaceutically acceptable carrier.

Use of one or more of 6-keto-prostaglandin F1a, prostaglandin A1, E6berbamine, juglone, GW-5074, rottlerin, cefixime, idarubicin,doxorubicin, methenamine, Congo red, sodium ferric gluconate, ferroussulfate, aurothioglucose, Evans blue, closantel, cinchonine sulfate,hexadimethrine bromide, indigotindisulfonate, or protamine chloride, orany combination thereof in the treatment of cancer is further provided.Use of one or more of 6-keto-prostaglandin F1a, prostaglandin A1, E6berbamine, juglone, GW-5074, rottlerin, cefixime, idarubicin,doxorubicin, methenamine, Congo red, sodium ferric gluconate, ferroussulfate, aurothioglucose, Evans blue, closantel, cinchonine sulfate,hexadimethrine bromide, indigotindisulfonate, or protamine chloride, orany combination thereof in the treatment of melanoma, colon cancer,recto-sigmoid colon cancer, prostate cancer, breast cancer, ovariancancer, pancreatic cancer, lung cancer, and/or brain cancer, includingglioblastoma, is further provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Western blot analysis of TDG in a portion of a panel of60 cancer cell lines. +/+ and −/− are the positive and negative controllysates from wild type and Tdg-null mouse embryo fibroblasts (MEFs). 293is an additional positive control.

FIG. 2 shows down-regulation of TDG in Mel501 cells.

FIG. 3 shows TDG down-regulation induces morphological changes in Mel501cells.

FIG. 4 shows expression of Tuj1 (stained spindle shapes), adifferentiated neuron-specific cell marker, in parental (right panels)and shC8 Mel501 (left panels). Nuclei were stained with DAPI.

FIG. 5 shows TDG down-regulation induces morphological changes in Mullcells.

FIG. 6 shows expression of Tuj1 (stained spindle shapes), adifferentiated neuron-specific cell marker, in parental (right panels)and shC8 MULL cells (left panels). Nuclei were stained with DAPI.

FIG. 7 shows reduced proliferation of TDG knock-out mouse embryofibroblast lines (MEFs, indicated as −/−), in comparison to wild typeand heterozygous MEFs (indicated as +1+ and +/−, respectively).

FIG. 8 shows the morphology of TDG wild type (+1+) and knock-out (−/−)MEFs.

FIG. 9 shows the total process length in parental Mel501 cells, shC8Mel501 cells and pLKO Mel501 cells.

FIG. 10 shows a quantitation of cellular processes in parental Mel501cells, shC8 Mel501 cells and pLKO Mel501 cells.

FIG. 11 shows a G2-M phase cell cycle arrest in TDG-downregulated MEL501cells.

FIG. 12 shows a G2-M phase cell cycle arrest in TDG-downregulated Mullcells.

FIG. 13 shows an S phase cell cycle arrest in TDG-downregulated SK28cells.

FIG. 14 shows a staining with an antibody against CENPF ofTDG-downregulated MEL501 cells.

FIG. 15 shows multinucleated MEL501 cells following downregulation ofTDG.

FIG. 16 shows reduced staining of melanocytic markers MelanA/Mart1 andTyrosinase in TDG-downregulated MEL501 cells.

FIG. 17 shows reduced MITF expression in TDG-downregulated melanoma celllines.

FIG. 18 shows that TDG downregulation inhibits the tumorigenic potentialof SK28 melanoma cells. Growth curves refer to two SCID mice injectedwith pLKO vector-infected (left flank) or shTDG C8-infected (rightflank) SK28 cells.

FIG. 19 shows the analysis of TDG activity using qPCR-based assay andidentification of candidate inhibitors. (A) Schematic of the molecularbeacon assay for G:T repair. (B) Dose dependent inhibition of juglone, acandidate TDG inhibitor at 0 (blue), 5 (teal), 50 (yellow) and 500(fuchsia) μg/ml. (C) A conventional glycosylase assay confirmsinhibition of TDG activity; inhibitors were tested at 100 nM, 10 μM and1 mM; compound 1, cefixime, is more potent than compound 2, closantel,in this assay.

FIG. 20 shows a schematic of the central role of TDG in DNAdemethylation pathways: the deamination (left),hydroxylation-deamination (center) and deamination-independent (right)pathways are shown. 5mC: 5-methylcytosine; 5hmC:5-hydroxymethylcytosine; T: thymine; 5hmU: 5-hydroxymethyluracil; 5fC:5-formylcytosine; 5caC: 5-carboxylcytosine; AP site:apurinic/apyrimidinic site; C: cytosine.

FIG. 21 shows elevated 5-carboxylcytosine levels associated withtargeted inactivation or downregulation of TDG. (A) Decreasing dilutionsof genomic DNA from embryos of the indicated Tdg genotype were blottedand detected with antibody anti-5caC. (B) Immuno-fluorescencedocumenting elevated levels of 5caC in SK28 melanoma cell line infectedwith sh lentivirus against TDG (C8) or vector control (pLKO).

FIG. 22 shows elevated 5-carboxylcytosine levels associated withtreatment of SK28 melanoma cells with the candidate TDG inhibitor,juglone.

FIG. 23 shows elevated 5-carboxylcytosine levels associated withtreatment of SK28 melanoma cells with the candidate TDG inhibitor,closantel.

FIG. 24 shows inhibition of TDG glycosylase activity in vitro byjuglone, and reduction of SK28 cell viability (MTS assay) andcolony-forming ability (clonogenic assay) upon treatment with juglone.

FIG. 25 shows inhibition of TDG glycosylase activity in vitro byclosantel, and reduction of SK28 cell viability (MTS assay) andcolony-forming ability (clonogenic assay) upon treatment with closantel.

FIG. 26 shows a reduction of clonogenic capacity of SK28 MEL melanomacells by juglone, closantel, and cefixime.

FIG. 27 shows that TDG downregulation leads to RAD51 activation, asassessed by the formation of RAD51 foci, in the absence of gamma-H2AXactivation, a marker of DNA damage response.

FIG. 28 shows highly elevated 5-carboxylcytosine levels associated withtemozolomide treatment combined with downregulation of TDG, suggesting asynergistic effect.

FIG. 29 shows highly elevated 5-carboxylcytosine levels associated withtemozolomide treatment combined with treatment of the cells withcandidate inhibitors of TDG, juglone and closantel, suggesting asynergistic effect.

FIG. 30 shows reduction of SK28 cell viability (MTS assay) whencisplatin treatment is combined with TDG downregulation (C8).

FIG. 31 shows elevated 5caC levels when cells are treated withvincristine even in the absence of TDG downregulation (pLKO); 5caClevels are further increased when vincristine treatment is combined withTDG downregulation (C8).

FIG. 32 shows the cell index from HCT-116 (colon cancer) cells infectedwith an empty vector or C8 lentivirus encoding shRNA for TDG knockdown.

FIG. 33 shows the cell index from A549 (lung cancer) cells infected withan empty vector or C8 lentivirus encoding shRNA for TDG knockdown.

FIG. 34 shows the cell index from PC-3 (prostate cancer) cells infectedwith an empty vector or C8 lentivirus encoding shRNA for TDG knockdown.

FIG. 35 shows the cell index from U251 (glioblastoma/brain cancer) cellsinfected with an empty vector or C8 lentivirus encoding shRNA for TDGknockdown.

FIG. 36 shows the cell index from HT-29 (recto-sigmoid colon cancer)cells infected with an empty vector or C8 lentivirus encoding shRNA forTDG knockdown.

FIG. 37 shows the cell index from MDA-MB-435 (breast cancer) cellsinfected with an empty vector or C8 lentivirus encoding shRNA for TDGknockdown.

FIG. 38 shows the cell index from SK28 (melanoma) cells infected with anempty vector or C8 lentivirus encoding shRNA for TDG knockdown.

FIG. 39 shows the cell index from SK28 melanoma) cells infected (secondinfection) with an empty vector or C8 lentivirus encoding shRNA for TDGknockdown.

FIG. 40 shows the cell index from MCF-7 (breast cancer) cells infectedwith an empty vector or C8 lentivirus encoding shRNA for TDG knockdown.

FIG. 41 shows the cell index from NCI-H23 (lung cancer) cells infectedwith an empty vector or C8 lentivirus encoding shRNA for TDG knockdown.

FIG. 42 shows morphologic changes in PC-3 and LNCap prostate cancercells from TDG knockdown.

FIG. 43 shows TDG inhibitors induce the killing of prostate cancercells.

FIG. 44 shows that SK28 melanoma cells senesce upon TDG knockdown.

FIG. 45 shows increased 5-carboxylcytosine (5caC) levels in the bonemarrow of myelodysplastic syndrome patients.

FIG. 46 shows immunohistochemistry detection of TDG expression and 5caCexpression levels.

DETAILED DESCRIPTION OF THE INVENTION

Various terms relating to aspects of the present invention are usedthroughout the specification and claims. Such terms are to be giventheir ordinary meaning in the art, unless otherwise indicated. Otherspecifically defined terms are to be construed in a manner consistentwith the definition provided herein.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless expressly stated otherwise.

Knockdown includes the reduced expression of a gene. A knockdowntypically has at least about a 20% reduction in expression, preferablyhas at least about a 50% reduction in expression, and more preferablyhas at least about a 75% reduction in expression, and in some aspectshas at least about an 80% to about an 85% reduction in expression, atleast about an 85% to about a 90% reduction in expression, or about an80% to about a 90% reduction in expression, and in some aspects has agreater than 90% reduction in expression, or a greater than 95%reduction in expression.

Transforming or transfecting a cell includes the introduction ofexogenous or heterologous nucleic acid molecules into the cell. Cellsmay be stably or transiently transformed or transfected.

Nucleic acid molecules include any chain of at least two nucleotides,which may be unmodified or modified RNA or DNA, hybrids of RNA and DNA,and may be single, double, or triple stranded.

Expression of a nucleic acid molecule comprises the biosynthesis of agene product. Expression includes the transcription of a gene into RNA,the translation of RNA into a protein or polypeptide, and all naturallyoccurring post-transcriptional and post-translational modificationsthereof.

Inhibiting includes reducing, decreasing, blocking, preventing,delaying, inactivating, desensitizing, stopping, knocking down (e.g.,knockdown), and/or downregulating the biologic activity or expression ofa gene, molecule or pathway of interest or cell growth or proliferation.

It has been observed in accordance with the invention that inhibition ofthe DNA base excision repair enzyme thymine DNA glycosylase (TDG)arrests the growth of cancer cells, including melanoma cells andprostate cancer cells, and induces differentiation and/or senescence ofcancer cells, including melanoma cells and prostate cancer ascharacterized by morphologic changes and phenotypes characteristic ofnon-cancerous cells. The inhibition of TDG produced similar results inmelanoma cells that express high and intermediate levels of TDG. It wasalso observed that inhibition of TDG in melanoma cells, colon cancercells, recto-sigmoid colon cancer cells, prostate cancer cells, breastcancer cells, ovarian cancer cells, pancreatic cancer cells, lung cancercells, and/or brain cancer cells, including glioblastoma cells inhibitsproliferation of such cells. These observations indicate TDG may betargeted in in various types of cancers.

For melanoma, it appeared that some melanoma cells that express lowlevels of TDG have tumor forming ability when injected into recipientmice in xenotransplantation experiments. It was initially believed thatreducing the levels of TDG in melanoma cells that carry high orintermediate levels of TDG expression, would increase their tumorforming ability. Surprisingly, reducing levels of TDG in melanoma cellswith relatively normal or intermediate expression of TDG resulted in asignificant inhibition of cell growth and the differentiation of suchcells toward a reversion to a healthy, non-cancerous phenotype andmorphology.

Accordingly, the invention features methods for inhibiting the growthand/or proliferation of cancer cells in which TDG is expressed, and/orfor inducing differentiation of TDG-expressing cells into non-cancerouscells, and/or for inducing senescence in TDG-expressing cells. Ingeneral, the methods comprise inhibiting the expression or biologicactivity of TDG in the cells. Any of the methods of the invention may becarried out in vivo, ex vivo, in vitro, or in situ.

The invention also features compositions for inhibiting the growthand/or proliferation of premalignant and/or cancer cells, and/or forinducing differentiation of premalignant and/or cancer cells intonon-cancerous cells or healthy cells, and/or for inducing senescence inpremalignant and/or cancer cells. Such compositions comprise any of6-keto-prostaglandin F1a, prostaglandin A1, E6 berbamine, juglone,GW-5074, rottlerin, cefixime, idarubicin, doxorubicin, methenamine,Congo red, sodium ferric gluconate, ferrous sulfate, aurothioglucose,Evans blue, closantel, cinchonine sulfate, hexadimethrine bromide,indigotindisulfonate, protamine chloride (e.g., SEQ ID NO: 8), or anypharmaceutically acceptable salt thereof, or any combination thereof.The compositions comprise a carrier such as a pharmaceuticallyacceptable carrier. The amount of the agent in the composition may be anamount effective to inhibit the growth and/or proliferation of cancercells, and/or to induce differentiation of into non-cancerous cells,and/or to induce senescence in the cells. The amount of the agent may betailored to the particular cancer, or to premalignant versus cancerouscells. The particular cancer (or premalignant state thereof) cells maycomprise melanoma cells, colon cancer cells, recto-sigmoid colon cancercells, prostate cancer cells, pancreatic cancer cells, ovarian cancercells, breast cancer cells, lung cancer cells, and/or brain cancercells, including glioblastoma cells.

Inhibiting the expression of TDG may comprise transfecting the cancer orpremalignant cell with a nucleic acid molecule that interferes with theexpression of TDG in the cell. For example, nucleic acid-basedinterference with TDG expression may take advantage of RNA interference.

RNA interference (RNAi) is a mechanism of post-transcriptional genesilencing mediated by double-stranded RNA (dsRNA), which is distinctfrom antisense and ribozyme-based approaches. RNA interference may beeffectuated, for example, by administering a nucleic acid (e.g., dsRNA)that hybridizes under stringent conditions to the gene encoding thymineDNA glycosylase (including mRNA encoding thymine DNA glycosylase),thereby attenuating its expression. RNA interference provides shRNA orsiRNA that comprise multiple sequences that target one or more regionsof the target gene. dsRNA molecules (shRNA or siRNA) are believed todirect sequence-specific degradation of mRNA in cells of various typesafter first undergoing processing by an RNase III-like enzyme calledDICER into smaller dsRNA molecules comprised of two 21 nucleotide (nt)strands, each of which has a 5′ phosphate group and a 3′ hydroxyl, andincludes a 19 nt region precisely complementary with the other strand,so that there is a 19 nt duplex region flanked by 2 nt-3′ overhangs.RNAi is thus mediated by short interfering RNAs (siRNA), which typicallycomprise a double-stranded region approximately 19 nucleotides in lengthwith 1-2 nucleotide 3′ overhangs on each strand, resulting in a totallength of between approximately 21 and 23 nucleotides. In mammaliancells, dsRNA longer than approximately 30 nucleotides typically inducesnonspecific mRNA degradation via the interferon response. However, thepresence of siRNA in mammalian cells, rather than inducing theinterferon response, results in sequence-specific gene silencing.

Viral vectors or DNA vectors encode short hairpin RNA (shRNA) which areprocessed in the cell cytoplasm to short interfering RNA (siRNA). Ingeneral, a short, interfering RNA (siRNA) comprises an RNA duplex thatis preferably approximately 19 basepairs long and optionally furthercomprises one or two single-stranded overhangs or loops. A siRNA maycomprise two RNA strands hybridized together, or may alternativelycomprise a single RNA strand that includes a self-hybridizing portion.siRNAs may include one or more free strand ends, which may includephosphate and/or hydroxyl groups. siRNAs typically include a portionthat hybridizes under stringent conditions with a target transcript. Onestrand of the siRNA (or, the self-hybridizing portion of the siRNA) istypically precisely complementary with a region of the target transcript(e.g., thymine DNA glycosylase transcript), meaning that the siRNAhybridizes to the target transcript without a single mismatch. Inaspects in which perfect complementarity is not achieved, it isgenerally preferred that any mismatches be located at or near the siRNAtermini.

siRNAs have been shown to downregulate gene expression when transferredinto mammalian cells by such methods as transfection, electroporation,cationic liposome-mediated transfection, or microinjection, or whenexpressed in cells via any of a variety of plasmid-based approaches. ThesiRNA may comprise two individual nucleic acid strands or of a singlestrand with a self-complementary region capable of forming a hairpin(stem-loop) structure. A number of variations in structure, length,number of mismatches, size of loop, identity of nucleotides inoverhangs, etc., are consistent with effective siRNA-triggered genesilencing. While not wishing to be bound by any theory, it is believedthat intracellular processing (e.g., by DICER) of a variety of differentprecursors results in production of siRNA capable of effectivelymediating gene silencing. Generally, it is preferred to target exonsrather than introns, and it may also be preferable to select sequencescomplementary to regions within the 3′ portion of the target transcript.Generally it is preferred to select sequences that contain anapproximately equimolar ratio of the different nucleotides and to avoidstretches in which a single residue is repeated multiple times.

siRNAs may thus comprise RNA molecules having a double-stranded regionapproximately 19 nucleotides in length with 1-2 nucleotide 3′ overhangson each strand, resulting in a total length of between approximately 21and 23 nucleotides. siRNAs also include various RNA structures that maybe processed in vivo to generate such molecules. Such structures includeRNA strands containing two complementary elements that hybridize to oneanother to form a stem, a loop, and optionally an overhang, preferably a3′ overhang. Preferably, the stem is approximately 19 bp long, the loopis about 1-20, more preferably about 4-10, and most preferably about 6-8nt long and/or the overhang is about 1-20, and more preferably about2-15 nt long. In certain aspects, the stem is minimally 19 nucleotidesin length and may be up to approximately 29 nucleotides in length. Loopsof 4 nucleotides or greater are less likely subject to stericconstraints than are shorter loops and therefore may be preferred. Theoverhang may include a 5′ phosphate and a 3′ hydroxyl. The overhang may,but need not comprise a plurality of U residues, e.g., between 1 and 5 Uresidues. Classical siRNAs as described above trigger degradation ofmRNAs to which they are targeted, thereby also reducing the rate ofprotein synthesis. In addition to siRNAs that act via the classicalpathway, certain siRNAs that bind to the 3′ UTR of a template transcriptmay inhibit expression of a protein encoded by the template transcriptby a mechanism related to but distinct from classic RNA interference,e.g., by reducing translation of the transcript rather than decreasingits stability. Such RNAs are referred to as microRNAs (miRNAs) and aretypically between approximately 20 and 26 nucleotides in length, e.g.,22 nt in length. It is believed that they are derived from largerprecursors known as small temporal RNAs (stRNAs) or mRNA precursors,which are typically approximately 70 nt long with an approximately 4-15nt loop. Endogenous RNAs of this type have been identified in a numberof organisms including mammals, suggesting that this mechanism ofpost-transcriptional gene silencing may be widespread. MicroRNAs havebeen shown to block translation of target transcripts containing targetsites.

siRNAs such as naturally occurring or artificial (i.e., designed byhumans) mRNAs that bind within the 3′ UTR (or elsewhere in a targettranscript) and inhibit translation may tolerate a larger number ofmismatches in the siRNA/template duplex, and particularly may toleratemismatches within the central region of the duplex. In fact, there isevidence that some mismatches may be desirable or required as naturallyoccurring stRNAs frequently exhibit such mismatches as do mRNAs thathave been shown to inhibit translation in vitro. For example, whenhybridized with the target transcript such siRNAs frequently include twostretches of perfect complementarity separated by a region of mismatch.A variety of structures are possible. For example, the mRNA may includemultiple areas of nonidentity (mismatch). The areas of nonidentity(mismatch) need not be symmetrical in the sense that both the target(e.g., thymine DNA glycosylase) and the mRNA include nonpairednucleotides. Typically the stretches of perfect complementarity are atleast 5 nucleotides in length, e.g., 6, 7, or more nucleotides inlength, while the regions of mismatch may be, for example, 1, 2, 3, or 4nucleotides in length.

Hairpin structures designed to mimic siRNAs and mRNA precursors areprocessed intracellularly into molecules capable of reducing orinhibiting expression of target transcripts (e.g., thymine DNAglycosylase). These hairpin structures, which are based on classicalsiRNAs consisting of two RNA strands forming a 19 bp duplex structureare classified as class I or class II hairpins. Class I hairpinsincorporate a loop at the 5′ or 3′ end of the antisense siRNA strand(i.e., the strand complementary to the target transcript whoseinhibition is desired) but are otherwise identical to classical siRNAs.Class II hairpins resemble mRNA precursors in that they include a 19 ntduplex region and a loop at either the 3′ or 5′ end of the antisensestrand of the duplex in addition to one or more nucleotide mismatches inthe stem. These molecules are processed intracellularly into small RNAduplex structures capable of mediating silencing. They appear to exerttheir effects through degradation of the target mRNA rather than throughtranslational repression as is thought to be the case for naturallyoccurring mRNAs and stRNAs.

Thus, a diverse set of RNA molecules containing duplex structures isable to mediate silencing through various mechanisms. Any such RNA, oneportion of which binds to a target transcript (e.g., thymine DNAglycosylase) and reduces its expression, whether by triggeringdegradation, by inhibiting translation, or by other means, may beconsidered an siRNA, and any structure that generates such an siRNA(i.e., serves as a precursor to the RNA) is useful.

A further method of RNA interference is the use of short hairpin RNAs(shRNA). A plasmid containing a DNA sequence encoding for a particulardesired siRNA sequence is delivered into a target cell (e.g., melanomacells, colon cancer cells, recto-sigmoid colon cancer cells, prostatecancer cells, pancreatic cancer cells, ovarian cancer cells, breastcancer cells, lung cancer cells, and/or brain cancer cells, includingglioblastoma cells) via transfection or virally-mediated infection. Oncein the cell, the DNA sequence is continuously transcribed into RNAmolecules that loop back on themselves and form hairpin structuresthrough intramolecular base pairing. These hairpin structures, onceprocessed by the cell, are equivalent to transfected siRNA molecules andare used by the cell to mediate RNAi of the desired protein. The use ofshRNA has an advantage over siRNA transfection as the former can lead tostable, long-term inhibition of protein expression. Inhibition ofprotein expression by transfected siRNAs is a transient phenomenon thatdoes not occur for times periods longer than several days. In somecases, though, this may be preferable and desired. In cases where longerperiods of protein inhibition are necessary, shRNA mediated inhibitionis preferable. The use of shRNA is preferred for some aspects of theinvention. Typically, siRNA-encoding vectors are constructs comprising apromoter, a sequence of the target gene to be silenced in the senseorientation, a spacer, the antisense of the target gene sequence, and aterminator.

Inhibition of the expression of thymine DNA glycosylase can also beeffectuated by other means that are known and readily practiced in theart. For example, antisense nucleic acids can be used. Antisense RNAtranscripts have a base sequence complementary to part or all of anyother RNA transcript in the same cell. Such transcripts modulate geneexpression through a variety of mechanisms including the modulation ofRNA splicing, the modulation of RNA transport and the modulation of thetranslation of mRNA. Accordingly, in certain aspects, inhibition of theexpression of thymine DNA glycosylase in a cancer cell can beaccomplished by expressing an antisense nucleic acid molecule in thecancer cell. The cancer cell may comprise one or more of melanoma cells,colon cancer cells, recto-sigmoid colon cancer cells, prostate cancercells, pancreatic cancer cells, ovarian cancer cells, breast cancercells, lung cancer cells, and/or brain cancer cells, includingglioblastoma cells.

Antisense nucleic acids are generally single-stranded nucleic acids(DNA, RNA, modified DNA, or modified RNA) complementary to a portion ofa target nucleic acid (e.g., an mRNA transcript) and therefore able tobind to the target to form a duplex. Typically, they areoligonucleotides that range from 15 to 35 nucleotides in length but mayrange from 10 up to approximately 50 nucleotides in length. Bindingtypically reduces or inhibits the expression of the target nucleic acid,such as the gene encoding the target signal protein. For example,antisense oligonucleotides may block transcription when bound to genomicDNA, inhibit translation when bound to mRNA, and/or lead to degradationof the nucleic acid. Inhibition of the expression of thymine DNAglycosylase can be achieved by the administration of antisense nucleicacids comprising sequences complementary to those of the mRNA thatencodes thymine DNA glycosylase.

Antisense oligonucleotides can be synthesized with a base sequence thatis complementary to a portion of any RNA transcript in the cancer cell.Antisense oligonucleotides can modulate gene expression through avariety of mechanisms including the modulation of RNA splicing, themodulation of RNA transport and the modulation of the translation ofmRNA. Various properties of antisense oligonucleotides includingstability, toxicity, tissue distribution, and cellular uptake andbinding affinity may be altered through chemical modifications including(i) replacement of the phosphodiester backbone (e.g., peptide nucleicacid, phosphorothioate oligonucleotides, and phosphoramidateoligonucleotides), (ii) modification of the sugar base (e.g.,2′-O-propylribose and 2′-methoxyethoxyribose), and (iii) modification ofthe nucleoside (e.g., C-5 propynyl U, C-5 thiazole U, and phenoxazineC).

Inhibition of thymine DNA glycosylase can also be effectuated by use ofribozymes. Certain nucleic acid molecules referred to as ribozymes ordeoxyribozymes have been shown to catalyze the sequence-specificcleavage of RNA molecules. The cleavage site is determined bycomplementary pairing of nucleotides in the RNA or DNA enzyme withnucleotides in the target RNA. Thus, RNA and DNA enzymes can be designedto cleave to any RNA molecule, thereby increasing its rate ofdegradation.

In some aspects, the cancer cells can be specifically transformed withtranscription-silencing nucleic acids such as shRNA or siRNA, or can betransformed with vectors encoding such nucleic acids such that the cellexpresses the inhibitory nucleic acid molecules. Transfection of thecancer cells can be carried out according to any means suitable in theart.

A cancer cell can be transfected with such nucleic acid moleculesaccording to any means available in the art such as those described orexemplified herein. It is preferred that the cancer cells are stablytransformed with a vector comprising a nucleic acid sequence encodingsuch regulatory nucleic acid molecules, although transientlytransformations are suitable. Any vector suitable for transformation ofthe particular cell of interest can be used. In preferred embodiments,the vector is a viral vector. In some embodiments, the viral vector is alentivirus vector.

In some preferred aspects, the nucleic acid molecule is a siRNA thatspecifically hybridizes under stringent conditions to mRNA encodingthymine DNA glycosylase. In some preferred aspects, the nucleic acidmolecule is a shRNA that specifically hybridizes under stringentconditions to mRNA encoding thymine DNA glycosylase. The shRNA maycomprise the nucleic acid sequence of SEQ ID NO: 3 or the nucleic acidsequence of SEQ ID NO: 4. The shRNA may hybridize to a nucleic acidencoding thymine DNA glycosylase including the nucleic acid sequence ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 6. Preferably,the thymine DNA glycosylase is human thymine DNA glycosylase (SEQ ID NO:7).

Inhibiting the biologic activity of TDG may comprise contacting the cellwith an effective amount of an agent that inhibits the biologic activityof TDG. The agent may comprise an organic or inorganic chemical(including a composition comprising such an organic or inorganicchemical, including a small molecule, and a carrier such as apharmaceutically acceptable carrier) that inhibits the biologic activityof TDG. The agent may comprise a biomolecule, including an antibody thatspecifically binds to TDG, or a polypeptide.

Biologic activity of TDG includes DNA/thymine glycosylase activity andexcision repair of thymine and uracil mismatches, including G/T, G/U,C/T, and T/T mismatches, as well as repair of hydroxymethyluracil,formylcytosine and carboxylcytosine opposite G. Biologic activity alsoincludes transcriptional co-activator activity. In some aspects, it maybe preferable to selectively inhibit glycosylase activity, for example,while retaining transcriptional co-activator activity.

The agent may comprise one or more of 6-keto-prostaglandin F1a,prostaglandin A1, E6 berbamine, juglone, GW-5074, rottlerin, cefixime,idarubicin, doxorubicin, methenamine, Congo red, sodium ferricgluconate, ferrous sulfate, aurothioglucose, Evans blue, closantel,cinchonine sulfate, hexadimethrine bromide, indigotindisulfonate, andprotamine chloride, or any combination thereof. In some preferredaspects, the agent comprises juglone. In some preferred aspects, theagent comprises cefixime. In some preferred aspects, the agent comprisesclosantel. Protamine may comprise the amino acid sequence of SEQ ID NO:8. Any of the agents may comprise a pharmaceutically acceptable saltthereof. Any of these agents may be comprised in a compositioncomprising the agent and a pharmaceutically acceptable carrier. Suchcompositions are within the scope of the invention.

Pharmaceutically acceptable salts may be acid or base salts.Non-limiting examples of pharmaceutically acceptable salts includesulfates, methosulfates, methanesulfates, pyrosulfates, bisulfates,sulfites, bisulfites, nitrates, besylates, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, dioates, benzoates, chlorobenzoates,methylbenzoates, dinitromenzoates, hydroxybenzoates, methoxybenzoates,phthalates, sulfonates, toluenesulfonates, xylenesulfonates,pheylacetates, phenylpropionates, phenylbutyrates, citrates, lactates,γ-hydroxybutyrates, glycollates, tartrates, methanesulfonates,propanesulfonates, mandelates, and other salts customarily used orotherwise FDA-approved.

The inhibition of the expression or biologic activity of TDG maysynergize with other agents for an enhanced cancer-treating effect.Thus, in some aspects, the method may further comprise contacting thecancer cell with one or more of a RAD51 inhibitor, a DNA alkylatingagent, temozolomide, dacarbazine, cisplatin, vincristine, or anycombination thereof. The cancer cell may comprise melanoma cells, coloncancer cells, recto-sigmoid colon cancer cells, prostate cancer cells,pancreatic cancer cells, ovarian cancer cells, breast cancer cells, lungcancer cells, and/or brain cancer cells, including glioblastoma cells.

In some aspects, the level of 5-carboxylcytosine may serve as abiomarker for efficacy of TDG inhibition. Preferably, elevated levels of5-carboxylcytosine indicate that TDG inhibition has occurred. Thus, themethods may optionally comprise, after inhibiting the expression orbiologic activity of TDG, detecting the level of 5-carboxylcytosine inthe cell, and if the level of 5-carboxylcytosine is not elevated,contacting the cell with a modulated, preferably increased, amount ofthe agent or with a different agent. These detecting and contactingsteps may be repeated any number of times sufficient in order to alterthe dosing of the agent, or at least to determine whether TDG inhibitionhas occurred. 5-carboxylcytosine levels reflecting TDG inhibitionpreferably are elevated over a baseline. The baseline preferably relatesback to a level of 5-carboxylcytosine in a cancer cell in which TDG hasnot been inhibited, or in which TDG has been inhibited at a low orinsufficient level. The agent is preferably the same agent initiallycontacted to the cells, but in some aspects, the agent is a differentagent. For example, if it is determined that the level of5-carboxylcytosine is not elevated, it may indicate that the first agenthas not inhibited the expression or biologic activity of TDG such that adifferent agent may be used.

Inhibiting the expression or biologic activity of TDG may cause thecancer cell to arrest growth in either the S phase of the cell cycle, orat the G2/M DNA damage checkpoint. It is preferred in some aspects thatthis arrest is sustained, such that the cell no longer grows. Sustenanceat either such point of the cell cycle may be maintained until the celldies, enters senescence, or differentiates into a non-malignant cell.

The invention also features methods for treating a cancer comprisingcells that express thymine DNA glycosylase, whether at high,intermediate, or low levels of expression, in a subject in need thereof.Subjects include, without limitation, mammals such as farm animals(e.g., horse, cow, sheep, pig), laboratory animals (e.g., mouse, rat,rabbit), companion animals (e.g., dog, cat), or non-human primates(e.g., new world monkey and old world monkey). In preferred aspects, thesubject is a human being. In general, the methods comprise inhibitingthe expression or inhibiting the biologic activity of TDG in the tumor,for example, by administering to the subject an effective amount of anagent that inhibits the expression of TDG and/or an agent that inhibitsthe biologic activity of TDG. The cancer/tumor cells may comprisemelanoma cells, colon cancer cells, recto-sigmoid colon cancer cells,prostate cancer cells, pancreatic cancer cells, ovarian cancer cells,breast cancer cells, lung cancer cells, and/or brain cancer cells,including glioblastoma cells

Administration may be directly to the tumor or indirectly to the tumor,for example, by administering the agent to the blood and allowing theagent to reach the tumor through the blood flow. The administration maycomprise active targeting of the agent to the tumor. The agent may beadministered systemically, or may be administered proximally or locallyto the tumor.

Inhibiting the expression of TDG may comprise transfecting a tumor cellin which TDG is expressed with a nucleic acid molecule that interfereswith the expression of TDG, including an RNA interference nucleic acidmolecule. Such transfection occurs following administration of thenucleic acid molecule to the subject. In some aspects, the nucleic acidmolecule is a siRNA that specifically hybridizes under stringentconditions to mRNA encoding TDG. In some preferred aspects, the nucleicacid molecule is a shRNA that specifically hybridizes under stringentconditions to mRNA encoding thymine DNA glycosylase. The shRNA maycomprise the nucleic acid sequence of SEQ ID NO: 3 or the nucleic acidsequence of SEQ ID NO: 4. The shRNA may hybridize to a nucleic acidencoding thymine DNA glycosylase including the nucleic acid sequence ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 6. Preferably,the thymine DNA glycosylase is human thymine DNA glycosylase (SEQ ID NO:7). Transforming a tumor cell may comprise infecting the tumor cell witha virus or other suitable delivery vehicle encoding the RNA interferencenucleic acid molecule. The virus may comprise a lentivirus.

Inhibiting the biologic activity of TDG may comprise contacting a tumorcell in which TDG is expressed with an effective amount of an agent thatinhibits the biologic activity of TDG. Such contacting occurs followingadministration of the agent to the subject. The agent may comprise anorganic or inorganic chemical (including a composition comprising suchan organic or inorganic chemical, including a small molecule, and acarrier such as a pharmaceutically acceptable carrier) that inhibits thebiologic activity of TDG. The agent may comprise a biomolecule,including an antibody that specifically binds to TDG, or a polypeptide.The agent may comprise one or more of 6-keto-prostaglandin F1a,prostaglandin A1, E6 berbamine, juglone, GW-5074, rottlerin, cefixime,idarubicin, doxorubicin, methenamine, Congo red, sodium ferricgluconate, ferrous sulfate, aurothioglucose, Evans blue, closantel,cinchonine sulfate, hexadimethrine bromide, indigotindisulfonate, andprotamine chloride, or any combination thereof. In some preferredaspects, the agent comprises juglone. In some preferred aspects, theagent comprises cefixime. In some preferred aspects, the agent comprisesclosantel.

Biologic activity of TDG includes DNA/thymine glycosylase activity andexcision repair of thymine and uracil mismatches, including G/T, G/U,C/T, and T/T mismatches, as well as repair of hydroxymethyluracil,formylcytosine and carboxylcytosine opposite G. Biologic activity alsoincludes transcriptional co-activator and transcriptional co-repressoractivity. In some aspects, it may be preferable to selectively inhibitglycosylase activity, for example, while retaining transcriptionalco-activator and transcriptional co-repressor activity, in the subject.

In some aspects, the method may further comprise administering to thesubject an effective amount of one or more of a RAD51 inhibitor, a DNAalkylating agent, temozolomide, dacarbazine, cisplatin, vincristine, orany combination thereof. Administration of any such agents orcombination may be prior to, substantially at the same time as, orfollowing administering to the subject an effective amount of an agentthat inhibits the expression of TDG and/or an agent that inhibits thebiologic activity of TDG.

In some aspects, the methods may optionally comprise detecting the levelof 5-carboxylcytosine in a sample of tumor tissue obtained from thesubject, for example, after administering the TDG expression- orbiologic activity-inhibiting agent and/or the RAD51 inhibitor, a DNAalkylating agent, temozolomide, dacarbazine, cisplatin, and/orvincristine, and if the level of 5-carboxylcytosine is not elevated inthe sample, administering to the subject a modulated, preferablyincreased, amount of the TDG expression- or biologic activity-inhibitingagent or administering to the subject a different TDG expression- orbiologic activity-inhibiting agent. Thus, for example, monitoring5-carboxylcytosine levels in the subject's tumor may serve as a way tomonitor treatment efficacy and make adjustments to the treatmentschedule in order to optimize treatment in the subject. Sampling ofpatient tumors and assessment of 5-carboxylcytosine levels may takeplace as frequently or infrequently as appropriate for guiding melanomatreatment in the subject. The tumor may comprise melanoma, colon cancer,recto-sigmoid colon cancer, prostate cancer, pancreatic cancer, ovariancancer, breast cancer, lung cancer, and/or brain cancer, includingglioblastoma.

5-carboxylcytosine elevation is believed to serve as a proxy foreffective TDG inhibition and, thus, effective cancer treatment,particularly for melanoma. 5-carboxylcytosine elevation also may be usedas a biomarker for certain cancers such as melanoma. Thus, in order todetermine whether 5-carboxylcytosine is elevated in a patient's tumors,it may be appropriate to determine a baseline level of5-carboxylcytosine in the tumor before initiating a TDG inhibitiontherapeutic regimen. Thus, in some aspects, the methods may optionallycomprise detecting the level of 5-carboxylcytosine in a sample of tumortissue obtained from the subject before administering the TDGexpression- or biologic activity-inhibiting agent and/or the RAD51inhibitor, a DNA alkylating agent, temozolomide, dacarbazine, cisplatin,and/or vincristine. In some alternative aspects, post-TDG inhibitoradministration-patient 5-carboxylcytosine levels may be compared againstpopulation-derived 5-carboxylcytosine baseline levels, rather than apatient-derived baseline level.

Detection of 5-carboxylcytosine may comprise a cancer diagnostic. Forexample, a method may comprise isolation of a tissue sample from asubject, and determination of whether 5-carboxylcytosine is expressed,or expressed at elevated levels indicative of a cancerous state may bemade. Determination of whether 5-carboxylcytosine is expressed, orexpressed at elevated levels in the tissue may indicate that the patienthas melanoma. colon cancer, recto-sigmoid colon cancer, prostate cancer,pancreatic cancer, ovarian cancer, breast cancer, lung cancer, and/orbrain cancer, including glioblastoma.

The invention also features kits. The kits may be used, for example, topractice any of the methods described or exemplified herein. In someaspects, a kit comprises a nucleic acid molecule that interferes withthe expression of thymine DNA glycosylase, and instructions for usingthe nucleic acid molecule in a method for inhibiting the growth ofmelanoma cells, and/or for inducing differentiation of cancer orpremalignant cells into non-cancerous cells, and/or for inducingsenescence in cancer or premalignant cells, and/or for treating cancerin a subject in need thereof. The cancer cells may comprise melanomacells, colon cancer cells, recto-sigmoid colon cancer cells, prostatecancer cells, pancreatic cancer cells, ovarian cancer cells, breastcancer cells, lung cancer cells, and/or brain cancer cells, includingglioblastoma cells. In some aspects, a kit comprises a nucleic acidmolecule that interferes with the expression of thymine DNA glycosylase,and instructions for using the nucleic acid molecule in a method fortreating cancer such as any method described or exemplified herein. Thenucleic acid molecule may be a siRNA and/or a shRNA that specificallyhybridizes under stringent conditions to mRNA encoding thymine DNAglycosylase. The shRNA may comprise the nucleic acid sequence of SEQ IDNO: 3 or the nucleic acid sequence of SEQ ID NO: 4. The shRNA mayhybridize to a nucleic acid encoding thymine DNA glycosylase includingthe nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5,or SEQ ID NO: 6. Preferably, the thymine DNA glycosylase is humanthymine DNA glycosylase (SEQ ID NO: 7). The non-cancerous cells maycomprise one or more of cells comprising a morphology characteristic ofmelanocytes, oligodendrocytes, astrocytes, or neurons. The non-cancerouscells may comprise one or more of melanocytes, oligodendrocytes,astrocytes, or neurons.

In some aspects, the kit comprises an agent that inhibits biologicactivity of thymine DNA glycosylase (TDG) and instructions for using thenucleic acid molecule in a method for inhibiting the growth of cancer orpremalignant cells, and/or for inducing differentiation of cancer cellsinto non-cancerous cells, and/or for inducing senescence in premalignantor cancer cells, and/or for treating cancer in a subject in needthereof. The biologic activity-inhibiting agent may comprise6-keto-prostaglandin F1a, prostaglandin A1, E6 berbamine, juglone,GW-5074, rottlerin, or any combination thereof. The biologicactivity-inhibiting agent may comprise cefixime, idarubicin,doxorubicin, methenamine, Congo red, sodium ferric gluconate, ferroussulfate, aurothioglucose, Evans blue, closantel, cinchonine sulfate,hexadimethrine bromide, indigotindisulfonate, protamine chloride, or anycombination thereof. Juglone, cefixime, and closantel are preferred.

In some aspects, the kit further comprises a RAD51 inhibitor,temozolomide, cisplatin, or vincristine, and instructions for using theRAD51 inhibitor, temozolomide, cisplatin, or vincristine in asynergistically-effective amount with the agent that inhibits theexpression of thymine DNA glycosylase or with the agent that inhibitsbiologic activity of thymine DNA glycosylase in a method for inhibitingthe growth of premalignant or cancer cells, and/or for inducingdifferentiation of cancer cells into non-cancerous cells, and/or forinducing senescence in premalignant or cells, and/or for treating cancerin a subject in need thereof. In some aspects, the kit further comprisesinstructions for determining the level of 5-carboxylcytosine in a sampleobtained from the tumor, and modulating, preferably increasing, theamount of the TDG expression- or biologic activity-inhibiting agent oradministering to the subject a different TDG expression- or biologicactivity-inhibiting agent, in order to enhance TDG inhibition.

The disclosure also features use of 6-keto-prostaglandin F1a,prostaglandin A1, E6 berbamine, juglone, GW-5074, rottlerin, cefixime,idarubicin, doxorubicin, methenamine, Congo red, sodium ferricgluconate, ferrous sulfate, aurothioglucose, Evans blue, closantel,cinchonine sulfate, hexadimethrine bromide, indigotindisulfonate, orprotamine chloride, or a pharmaceutically acceptable salt thereof, or acomposition thereof, or any combination thereof in the manufacture of amedicament for the treatment of cancer, including melanoma, lung cancer,breast cancer, colon cancer, recto-sigmoid colon cancer, prostatecancer, pancreatic cancer, ovarian cancer, brain cancer, and/orglioblastoma.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

Example 1 Reduced TDG Expression in Melanoma Cells

Because G:T and G:U repair systems are generally effective in protectingcells from spontaneous mutagenesis, it was hypothesized thatinactivating mutations of TDG may accelerate the accumulation of thesetypes of mutation in certain cancer genes. In preliminary evaluations,it was observed that TDG expression is frequently reduced or absent incertain cancer cell lines, particularly with respect to melanoma (FIG.1). From these results, it was hypothesized that reducing the levels ofTDG in melanoma cells would increase their tumor forming ability. As theExamples below illustrate, however, reducing TDG levels in melanomacells not only did not increase their tumor forming ability, it reducedtheir growth and induced differentiation toward a healthy phenotype.

Example 2 Knockdown of TDG in Melanoma Cells that Express High Levels ofTDG

The base excision repair thymine DNA glycosylase (TDG) has a dual rolein prevention of mutations that may originate from deamination of5-methylcytosine and in transcriptional regulation. Based on work on TDGknock-out mouse embryos, whose phenotypes suggested an involvement ofneural crest cells, the precursors of melanocytes, and the fact thatmelanoma cell lines were observed to have low levels of TDG proteins, itwas hypothesized that modulation of TDG levels may affect the biology ofmelanoma.

Initial experiments demonstrated that downregulation of TDG levels inMel501, a melanoma line characterized by high endogenous levels of TDG,caused reduced growth and induced characteristic morphological changes.As explained below, upon shTDG silencing, Mel501 cells lost the typicalspindle shape to present higher quantities of cellular processesresembling dendrites, a characteristic of melanocytes, oligodendrocytes,astrocytes, and neurons. Thus, it is believed that downregulation of TDGlevels, and perhaps even inhibition of its glycosylase activity, mayrepresent a valuable therapeutic opportunity in a fraction of melanomacases, exemplified by Mel501 cells, causing growth inhibition anddifferentiation.

Mel501 cell lines were infected with a sh lentivirus specific for TDG,named shC8, and in parallel, Mel501 cells were infected with a controllentivirus specific for green fluorescent protein, named shGFP. Stablecell lines expressing each lentivirus were selected using the Puromycinselectable marker. All the experiments were conducted in duplicate andperformed twice in order to further validate every result obtained withbiological duplicates. After 2 weeks of antibiotic selection, thedown-regulation of TDG by lentiviral vector shC8, but not shGFP, wasconfirmed by Western blot analysis of lysates from parental and infectedMel501 cells (FIG. 2).

A significant morphological change was observed in Mel501 cells, which,upon infection with the shC8 lentivirus downregulating TDG, lost thetypical spindle shape (FIG. 3, left) and assumed a morphologycharacteristic of melanocytes, oligodendrocytes, astrocytes, or neurons(FIG. 3, center). This effect was specific for TDG downregulation,because the changes induced by the shGFP control lentivirus were moresubtle, though some cells resembling astrocytes were observed upon shGFPinfection (FIG. 3, right). Initial evidence of neuronal differentiationwas obtained by showing that Mel501 cells with TDG downregulationexpress the neuronal marker Tuj1 (FIG. 4).

Example 3 Knockdown of TDG in Melanoma Cells that Express IntermediateLevels of TDG

In Example 2 above, the data show that downregulation of TDG levels inMel501, a melanoma cell line characterized by high endogenous levels ofTDG, caused reduced proliferation and induced characteristicmorphological changes such as the appearance of dendrites, which arecellular processes characteristic of melanocytes, oligodendrocytes,astrocytes, and neurons. Follow-up experiments were conducted in asecond melanoma cell line, which expresses intermediate levels of TDG,or in any event, lower levels of TDG relative to Mel501 cells. Theseexperiments, conducted in MULL cells, showed similar results to thoseobserved as part of the experiments of Example 2.

MULL cells were infected with a sh lentivirus specific for TDG, namedshC8, and parallel MULL cell cultures were infected with an empty vectorcontrol lentivirus, named shPLKO. Stable cell lines expressing eachlentivirus were selected using the Puromycin selectable marker. All ofthese experiments were conducted in duplicate and performed twice inorder to further validate every result obtained with biologicalduplicates.

A significant morphological change was observed in MULL cells uponinfection with the shC8 lentivirus downregulating TDG. Specifically,MULL cells lost the typical triangular, elongated, spindle shape (FIG.5, left), and each developed several dendritic processes, thus acquiringa morphology characteristic of melanocytes, oligodendrocytes, astrocytesor neurons (FIG. 5, center). This effect was specific for TDGdownregulation, because the changes induced by the shPLKO control emptylentivirus were more subtle, though some cells resembling astrocyteswere observed upon shPLKO infection (FIG. 5, right). Similar to theresults observed for experiments in Mel501 cells (Example 2), MULL cellswith TDG downregulation showed evidence of neuronal differentiation byexpressing the neuronal marker Tuj1 (FIG. 6).

Example 4 Targeted Inactivation of TDG is Associated with ReducedCellular Proliferation in Mouse Embryo Fibroblasts

As described in Examples 2 and 3 above, both Mel501 and MULL cellsshowed reduced proliferation upon downregulation of their endogenouslevels of TDG. A similar effect was noted in mouse embryo fibroblasts(MEFs) derived from mouse embryos with targeted inactivation (knock-out)of TDG. Compared to their wild type (no TDG knock-out) counterpart, TDGknock-out MEFs exhibited reduced proliferation (FIG. 7). This decreasedproliferation rate was associated with morphological changes (flattened,enlarged, elongated cytoplasm), resembling those of senescent cells(FIG. 8).

Example 5 Analysis of Cellular Processes

To quantify the effect of TDG downregulation on the differentiation oflentivirus-infected Mel501 cells, further analysis was conducted on thecellular processes resembling dendrites, since it is believed that theserepresent a significant feature of differentiation. Image analysis-basedapproaches similar to the ones used to quantify dendrite developmentduring neurogenesis were used. By using Image J software on microscopeimages of the different cultures, total length of processes per cell (inpixels) was measured, as well as the number of processes per cell. Theresults showed that Mel501 cells infected with the sh lentivirus C8directed against TDG mRNA exhibit an increase in both total length ofprocesses per cell (FIG. 9) and in the number of processes per cell(FIG. 10).

Example 6 Cell Cycle Arrest and Multinucleation

The foregoing examples indicate that downregulation of TDG inducesgrowth arrest in melanoma cell lines. Additional experiments furthercharacterized this growth arrest, and showed that downregulation of TDGinduces cell cycle arrest either in the G2-M phase (as an example,MEL501 or Mull melanoma cell lines) or S phase (as an example SK28melanoma cell lines) of the cell cycle, as shown by florescenceactivated cell sorting (FACS) (FIGS. 11-13). In addition, staining ofTDG-downregulated MEL501 cells with an antibody against CENPF indicatedthat these cells are arrested in either late S phase or G2 phase of thecell cycle (FIG. 14). Some MEL501, Mull and SK28 cells with TDGdownregulation escaped the cell cycle arrest and accumulated >4n DNAcontent, in agreement with the appearance of multinucleated cellsfollowing TDG downregulation (FIG. 15).

Example 7 Decrease of MITF Levels and Induction of Senescence

Downregulation of TDG is associated with increased expression of Tuj-1,a neuronal differentiation marker. It has now been observed that thelevels of melanocytic differentiation markers Tyrosinase andMelan-A/MART1 were reduced by TDG downregulation (FIG. 16). The maintranscriptional regulator of Tyrosinase and Melan-A/MART1 expression isthe Microphtalmia Transcription Factor (MITF). Accordingly, MITF levelswere assessed in melanoma cell lines with TDG downregulation.

In TDG-downregulated melanoma cells, a dramatic decrease of MITFexpression level was detected by Western blotting (FIG. 17). Withoutintending to be limited to any particular theory or mechanism of action,it is hypothesized that the morphological changes induced by TDGdownregulation may be a reflection not only of differentiation, but alsoof senescence, because MITF silencing has been shown to inducesenescence. Thus, it is believed that TDG inhibition may causesenescence of melanoma cells by decreasing MITF levels. It is believedthat the growth arrest may be related to the induction of senescence.

Example 8 Reduction of Tumor Formation in Xenotransplants

Cells with TDG downregulation are growth-arrested and exhibit reductionof viability. Therefore, it was hypothesized that their tumorigenicpotential should be compromised. Accordingly, the tumorigenicity of suchcells was evaluated in a xenotransplant assay. Cells were injectedsubcutaneously, in either flank, of two SCID mice: SK28 cells infectedwith the shRNA lentivirus against TDG or SK28 cells infected withcontrol pLKO lentivirus. Only the latter were able to form tumors,whereas the cells with TDG downregulation failed to form tumors (FIG.18).

Example 9 Identification of Candidate TDG Inhibitors

In order to identify inhibitors of TDG glycosylase activity, an in vitroassay that employs a molecular beacon, a hairpin-shaped oligonucleotidewith a G:T or G:U mismatch (substrate) and highly active preparations ofrecombinant TDG and recombinant AP endonuclease (APE) was employed. Inthe folded hairpin substrate, the fluorescence of 6-FAM, used asfluorescent label at the 5′ end, is quenched by a dabsyl “black hole”moiety at the 3′ end. Upon removal of the mismatched T or U by TDG, andincision of the resulting apurinic/apyrimidinic (AP site) by APE, ashort oligonucleotide containing 6-FAM was released. The resultingfluorescence was monitored by real-time qPCR over a 2-hour incubationperiod at 37° C., providing a sensitive and quantitative measurement ofrepair activity.

This assay was optimized for a 96-well and 384-well format. Uponscreening the ICCB known bioactive library (approximately 500 compounds)and the Johns Hopkins clinical compound library (approximately 1500drugs), eighteen and fourteen candidates, respectively (Tables 1 and 2)were identified. Some of these compounds confirmed TDG inhibition in astandard, radioactive-based glycosylase assay (FIG. 19).

TABLE 1 Candidate TDG inhibitors identified by screening the ICCBlibrary. 6-Keto-prostaglandin F1a7-[(1R,2S)-2-[(E,3S)-3-hydroxyoct-l-enyl]-5-oxocyclopent-3-en-1-yl]heptanoic acid

E6 Berbamine 6,6′,7-Trimethoxy-2,2′-dimethylberbaman-12- yl acetate

Prostaglandin A1 9-oxo-15S-hydroxy-prosta-10,13E-dien-1-oic acid

Juglone 5-hydroxynaphthoquinone

GW-5074 3-(3,5-Dibromo-4-hydroxybenzylidine-5-iodo-1,3-dihydro-indol-2-one

Rottlerin (E)-1-[6-[(3-acetyl-2,4,6-trihydroxy-5-methylphenyl)methyl]-5,7-dihydroxy-2,2-dimethylchromen-8-yl]-3-phenylprop-2-en-1- one

TABLE 2 Candidate TDG inhibitors identified by screening the JHCClibrary. Cefixime (6R,7R)-7-{[2-(2-amino- 1,3-thiazol- 4-yl)-2-(carboxymethoxyimino)- acetyl]amino}- 3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2- carboxylic acid

Idarubicin (1S,3S)-3-acetyl-3,5,12- trihydroxy- 6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl 3-amino- 2,3,6-trideoxo-α-L-lyxo- hexopyranoside

Doxorubicin (7S,9S)-7-[(2R,4S,5S,6S)- 4-amino-5-hydroxy-6-methyloxan-2-yl]oxy- 6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10- dihydro-7H-tetracene-5,12-dione

Methenamine (Hexamethylenetetramine)

Congo red disodium 4-amino-3-[4-[4- (1-amino- 4-sulfonato-naphthalen-2-yl)diazenylphenyl]- phenyl]diazenyl- naphthalene-1-sulfonate

Sodium ferric gluconate (Ferrlecit ®)

Ferrous sulfate

Aurothioglucose gold(I) (2S,3S,4R,5S)-3,4,5-trihydroxy-6-(hydroxymethyl)- oxane-2-thiolate

Evans blue tetrasodium (6E,6′E)-6,6-[(3,3′- dimethylbiphenyl-4,4′-diyl)di(1E)hydrazin-2-yl-1- ylidene]bis(4-amino-5-oxo-5,6-dihydronaphthalene-1,3- disulfonate)

Closantel 5′-Chloro-4′-(4-chloro-α- cyanobenzyl)-3,5-diiodo-2′-methylsalicylanilide, N- [5-Chloro-4- (4-chloro-α-cyanobenzyl)-2-methylphenyl]-2-hydroxy-3,5- diiodobenzamide

Cinchonine sulfate

Hexadimethrine bromide (Polybrene) 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, hexadimethrine bromide -

Indigotindisulfonate (Indigo Carmine) 3,3′-dioxo-2,2′-bis-indolyden-5,5′- disulfonic acid disodium salt

Protamine chloride, grade V MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR (SEQ IDNO: 8)

Example 10 Downregulation or Inhibition of TDG Causes Elevated Levels of5-Carboxylcytosine (5caC)

DNA modifying enzymes of the ten-eleven translocation (TET) family andbase excision repair DNA glycosylases are involved in DNA demethylation,an epigenetic de-modification associated with gene activation.Specifically, TET family proteins TET1, 2 and 3 are dioxygenases thatoxidize 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). TET proteinssubsequently convert the 5hmC to 5-formylcytosine (5fC) and5-carboxylcytosine (5caC), and TDG removes 5fC and 5caC opposite G.While potential accessory roles of the glycosylases MED1/MBD4 and SMUG1,and deaminases of the AID/APOBEC family cannot be ruled out completely,the bulk of the currently available data point to the TET-TDG axis as acentral component of the pathways mediating active cytosinedemethylation via conversion of 5mC to 5hmC, and then sequentially to5fC and 5caC (FIG. 20). By immunodot-blot of DNA extracted from cellsand immunofluorescence staining of cells, elevated levels of 5caC weredetected in embryos genetically deleted of TDG and cell linesdownregulated of TDG. Without intending to be limited to any particulartheory or mechanism of action, it is believed that inhibition or loss ofTDG alters the epigenome (FIG. 21).

Two compounds from the screen described in Example 9, juglone andclosantel, were found to increase 5caC staining in the nuclei of cellsin culture, confirming TDG inhibition (FIGS. 22-23).

Example 11 Two Putative TDG Inhibitors Reduce Cell Viability andClonogenic Capacity

Juglone, a quinone chemopreventive agent extracted from the blackwalnut, closantel, an anti-helminth drug, and cefixime, an antibioticfrom the cephalosporin family were found to reduce cell viability andclonogenic capacity of SK28 cells in a concentration-dependent fashion(FIG. 24-26). Tests were conducted in quadruplicate.

Example 12 Downregulation of TDG Causes the Appearance of RAD51 Foci

RAD51 is an important protein in the repair of DNA double strand breaksby homologous recombination. Specifically, RAD51 is involved in thesearch for homology, forming helical nucleoprotein filaments on DNA thatappear as “foci” upon staining with a specific antibody. It was observedthat cells with downregulation of TDG accumulate RAD51 foci (FIG. 27).It is believed that TDG downregulation/inhibition potentially maysynergize with RAD51 inhibitors for cancer treatment.

Example 13 Downregulation/Inhibition of TDG Synergizes with Temozolomide

Alkylating agents are a class of DNA damaging and anti-cancer drugswhose main mechanism of action consists in the alkylation of guanine inDNA to form 06-methylguanine. Two alkylating agents are used in theclinic, temozolomide and dacarbazine.

Treatment of cancer cells with temozolomide was observed to cause adramatic increase of 5caC levels when combined with TDG downregulation(FIG. 28) or inhibition (FIG. 29). This indicates that combinatorialtreatment of cancer cells with alkylating agents plus TDGdownregulation/inhibition can have a synergistic effect in killingcancer cells.

Example 14 Downregulation of TDG Synergizes with Cisplatin

Cisplatin is a chemotherapeutic agent that forms intra- and interstrandadducts. It was observed that treatment of cancer cells with cisplatincaused a reduction in viability when combined with TDG downregulation(FIG. 30).

Example 15 Downregulation of TDG Synergizes with Vincristine

Vincristin is an inhibitor of mitosis that arrests cells in metaphase;it is used as a chemotherapeutic agent for leukemia/lymphoma andmelanoma. It was observed that treatment of cancer cells withvincristine caused an increase of 5caC levels even in the absence of TDGdownregulation; however, 5caC levels were further increased whenvincristine treatment was combined with TDG downregulation (FIG. 31).This indicates that combinatorial treatment of cancer cells withvincristine plus TDG downregulation/inhibition can have a synergisticeffect in killing cancer cells.

Example 16 TDG Down-Regulation Suppresses Proliferation of MultipleCancer Cell Types

The effect of TDG down-regulation on the growth of various cancer celltypes was investigated using the xCELLigence® (Roche Diagnostics GmbH)real-time cell analyzer, a label-free, non-invasive method to monitoradherent cell behavior, including proliferation, spreading andcompound-mediated cytotoxicity. The system is based on detectingimpedance differences within an electrical circuit created inmicroelectrodes at the base of culture wells; these differences areconverted into a cell index (CI), a value that is influenced by avariety of factors, such as cell number, cell size and cell adhesion.Over an incubation time of 160 hours, a marked difference in cell indexwas detected for cancer cell pairs, infected with either empty vector orC8 lentivirus, indicating that their proliferation was suppressed by TDGdown-regulation (FIGS. 32-41). Cell lines from different cancer typesdecreased their proliferation upon TDG downregulation, including HCT-116(colon cancer), HT-29 (recto-sigmoid colon cancer), A549 and NCI-H23(lung cancer), PC3 (prostate cancer), U251 (glioblastoma/brain cancer),MDA-MB-435 and MCF-7 (breast cancer); this analysis also confirmed thatTDG downregulation decreased the proliferation of SK28 melanoma cells(FIG. 32-41). Similar data were obtained for pancreatic and ovariancancer cells (data not shown).

Example 17 TDG Down-Regulation Induces Morphological Changes in ProstateCancer Cells

The effect of TDG down-regulation on the morphology of prostate cancercells was tested. Much like what was observed in melanoma cell lines(FIG. 3, FIG. 5), both PC3 and LNCap cells developed numerous and longdendritic processes (FIG. 42). Without intending to be limited to anyparticular characterization, theory, or mechanism of action, it isbelieved that these changes, by analogy with the melanoma results, arelikely due to the induction of senescence; however, it is also believedthat it is possible that these changes may represent differentiation ofthe cells. These results were also observed in many other cancer celltypes upon TDG downregulation (data not shown) such that it is believedthat such morphologic changes may be a more generalized phenomenonbeyond melanoma, prostate, and other cancers tested in accordance withthis Example.

Example 18 Candidate TDG Inhibitors Induce Killing of Prostate CancerCells

The TDG inhibitors, juglone and closantel, were tested for their abilityto kill PC3 and LNCaP prostate cancer cells. Consistent with the shRNAresults, these compounds stopped proliferation of the prostate cancercells, as determined by MTS and clonogenic assay (FIG. 43).

Example 19 TDG Down-Regulation Induces Senescence in Melanoma

An important marker of senescence is the expression of endogenouslysosomal beta-galactosidase, which can be assayed by conducting achromogenic beta-galactosidase assay at pH 6.0. This assay is calledSenescence-associated beta-galactosidase staining (SA-β-gal). It wasobserved that SK28 melanoma cells are positive for SA-β-gal upon TDGdownregulation (FIG. 44). It is believed that these data support thehypothesis that TDG downregulation induces senescence.

Example 20 Elevated 5-Carboxylcytosine (5caC) as a Biomarker ofPre-Malignant Conditions and Cancer

It was hypothesized that some human premalignant and malignantconditions might be characterized by defects in TDG expression and/orbiological activity, which, in turn, would lead to elevated 5caC levels,based on the pathway in FIG. 20. Since levels of 5caC are very low orundetectable in normal cells, detection of 5caC would represent avaluable biomarker for premalignant conditions and cancer.

Myelodysplastic Syndrome (MDS) comprises a heterogeneous group ofdebilitating and malignant disorders of the hematopoietic tissue. Theyare characterized by uni- or multilineage dysplasia, ineffectivehematopoiesis, peripheral cytopenias and increased risk of evolutioninto overt acute myeloid leukemia. An immuno-dot blot procedure was usedin which serially diluted genomic DNA are blotted on a nitrocellulosemembrane and then detected with an antibody anti-5caC. Analysis of bonemarrow DNA samples from ˜30 MDS patients revealed increased levels of5caC in 8 cases (FIG. 45). FIG. 45 shows dilutions of genomic DNA (highto low from top to bottom) from bone marrow of MDS cases, blotted anddetected with antibody anti-5caC; arrows mark cases with elevated 5caC.Thus, elevated 5caC defines a subset of MDS cases.

For melanoma, immunohistochemistry (IHC) was used to detect expressionof TDG and levels of 5caC, using specific antibodies. The staining isvery specific and allows detection of TDG and 5caC levels (FIG. 46).FIG. 46 shows IHC staining of a melanoma with anti-TDG (left,DAB-positive nuclei) and anti-5caC (right, VIP-positive nuclei)antibodies. Areas of the tumor that lost TDG expression (red contour)have higher levels of 5caC. Notably, the areas of the tumor with reducedexpression of TDG and high levels of 5caC are morphologically distinctfrom areas of the tumor with expression of TDG retained and low levelsof 5caC. While the latter have melanoma cells with bigger nuclei and lowcell density, the former have melanoma cells with smaller nuclei andhigh cell density. Thus, without intending to be limited to anyparticular theory of mechanism of action, it is believed that reducedTDG and elevated 5caC may define melanomas with defined cytologicalfeatures that could lead to different clinico-pathologicalcharacteristics.

The invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

We claim:
 1. A method of treating a patient having melanoma, prostatecancer, colon cancer, or glioblastoma, comprising administering to theblood of the patient in need thereof an amount of aurothioglucoseeffective to inhibit TDG in cells of the melanoma, prostate cancer,colon cancer, or glioblastoma.
 2. The method according to claim 1,further comprising administering temozolomide to the patient.
 3. Themethod according to claim 1, further comprising administering cisplatinto the patient.
 4. The method according to claim 1, further comprisingadministering vincristine to the patient.
 5. The method according toclaim 1, further comprising administering a RAD51 inhibitor to thepatient.
 6. A method of treating a patient having prostate cancercomprising administering to the blood of the patient in need thereof aninhibitor of TDG in the amount effective to inhibit TDG in cells ofprostate cancer, wherein the TDG inhibitor comprises aurothioglucose. 7.The method according to claim 6, further comprising administeringtemozolomide to the patient.
 8. The method according to claim 6, furthercomprising administering cisplatin to the patient.
 9. The methodaccording to claim 6, further comprising administering vincristine tothe patient.
 10. The method according to claim 6, further comprisingadministering a RAD51 inhibitor to the patient.
 11. The method accordingto claim 1, wherein the patient has melanoma.
 12. The method accordingto claim 1, wherein the patient has glioblastoma.
 13. A method oftreating a patient having colon cancer comprising administering to theblood of the patient in need thereof an inhibitor of TDG in the amounteffective to inhibit TDG in cells of the colon cancer, wherein the TDGinhibitor comprises aurothioglucose.
 14. The method according to claim13, further comprising administering temozolomide to the patient. 15.The method according to claim 13, further comprising administeringcisplatin to the patient.
 16. The method according to claim 13, furthercomprising administering vincristine to the patient.
 17. The methodaccording to claim 13, further comprising administering a RAD51inhibitor to the patient.